The CAD protein is a multifunctional protein which carries out the first three steps of pyrimidine biosynthesis in mammalian cells. The goals of this proposal are to study the regulation and expression of the CAD protein and the organization and regulation of the CAD gene. The aims directed toward reaching these goals include i) analysis of CAD protein expression, synthesis, and turnover in hamster cell mutants; ii) DNA changes associated with mutants; iii) generation of new mutants; iv) expression of part of the hamster CAD gene in bacteria; and v) analysis and mapping of the human CAD gene. The approach involves using methods from somatic cell genetics, biochemistry, and molecular biology. Such techniques as polyacrylamide gel electrophoresis, immunoprecipitation, whole cell labeling, enzyme assays, recombinant DNA techniques, mutagenesis, and mamalian cell culture will be used. These studies will help develop a more complete picture of CAD regulation from gene to protein. Furthermore, this research may elucidate those changes in CAD expression associated with malignancy. Through a better understanding of a protein critical to cell multiplication, new methods for controlling cancer may, in time, be developed.